Apparatus and method for separation of liquid phases of different density and for fluorous phase organic syntheses

ABSTRACT

A simple, efficient apparatus and method for separating layers of immiscible or partially miscible liquids useful in methods of high-throughput combinatorial organic synthesis or parallel extraction of large libraries or megaarrays of organic compounds is disclosed. The apparatus and method are useful, whether as part of an automated, robotic or manual system for combinatorial organic synthesis or purification (extraction). In a preferred embodiment, an apparatus and method for separating layers of immiscible or partially miscible liquids compatible with microtiter plate type array(s) of reaction vessels is disclosed. Another application of centrifugation based liquid removal was found for washing the plates in biological assays or synthesis on modified substrates.

This application claims benefit of U.S. Provisional Application60/118,377 filed Jan. 29, 1999, which is expressly incorporated hereinby reference.

FIELD OF INVENTION

The present invention relates to the field of devices and methods forchemical synthesis, analysis, and biological screening. Moreparticularly, the present invention relates to a simple efficientapparatus and method for separation of immiscible or partially miscibleliquid phases of different density in high-throughput, organicsynthesis, or for removal of liquid from the vessels (washing). Thepresent invention is particularly applicable for high-throughputcombinatorial synthesis of organic molecules, whether as part of anautomated or a manual procedure.

BACKGROUND OF THE INVENTION

Solid phase synthesis of organic molecules is the method of choice forpreparation of libraries and compound megaarrays, which are currentlybeing applied for screening in the quest to find new drugs orpharmaceutical lead compounds, i.e., compounds which exhibit aparticular biological activity of pharmaceutical interest, and which canserve as a starting point for the selection and synthesis of a drugcompound, which in addition to the particular biological activity ofinterest has pharmacologic and toxicologic properties suitable foradministration to animals, including humans.

Fluorous synthesis is in its principle similar to solid phase synthesis.In fluorous synthesis the certain component of the reaction (startingmaterial, reagent, or product) is preferentially retained in thefluorine atoms containing phase due to its high content of fluorineatoms. Fluorous phase is usually the high density one and therefore itcan be separated as the lower phase in the multiphase system. Manualsynthesis requires repetitions of several relatively simple operationsof addition of reagents, incubation and separation of liquid phases.This character of the synthetic process renders it optimal forautomation.

Several designs of automated instruments for combinatorial synthesisutilizing solid phase synthesis have appeared in the patent andnon-patent literature. However, there is no instrument designed for thefluorous synthesis, since the simple principle of separation of phasesby filtration is not applicable.

The productivity of automated instruments can be dramatically improvedby use of disposable reaction vessels (such as multititer plates or testtube arrays) into which reagents are added by pipetting, or by directdelivery from storage containers. The optimal storage vehicle is asyringe-like apparatus of a material inert to the chemical reactants,etc., e.g., a glass syringe, allowing the storage of the solutionwithout any exposure to the atmosphere, and capable of serving as adelivery mechanism at the same time. See U.S. Pat. No. 6,045,755. Analternative technique based on the removal of upper layer of liquid bysuction from the surface above the separated layers is limited to thearrays of up to a hundred of suctions (For similar situation in solidphase synthesis see U.S. Pat. No. 6,045,755. The present application isan improvement upon U.S. Pat. Nos. 5,202,418, 5,338,831, 5,342,585, and6,045,755 which describe placement of resin in polypropylene meshpackets and removal of liquid through the openings of these packets, orremoval of the liquid from the pieces of porous textile-like material bycentrifugation, or removal of liquid phase from the solid phase bycentrifugation of tilted plates. Liquid removal by centrifugation wasdescribed and is the subject of several publications (see the book“Aspects of the Merrified Peptide Syntheses” by Christian Birr in theseries Reactivity and Structure Concepts in Organic Chemistry vol. 8, K.Hafner, J. -M. Lehn, C. W. Rees, P. von Rague, Schleyer, B. M. Trost, R.Zahradnik, Eds., Sringer-Verlag, Berlin, Heidelberg, N.Y. 1978, andGerman Patent Application P 20 17351.7, G. 70 13256.8, 1970, thesereferences describe the use of centrifugation for liquid removal fromslurry of solid phase particles in a concentrical vessel equipped with afiltration material in its perimeter and spun around its axis. See alsoW099/25470, hereby expressly incorporated by reference in its entirety.

None of the prior art contemplates the separation of two (or more)immiscible, or partially miscible liquids of different density byremoval of lighter layers of liquids by creation of “pockets” from whichmaterial cannot be removed by centrifugal force. This technique can beused in situations where multiplicity of products are to be extracted inparallel (e.g. in parallel purification of products of combinatorialsynthesis). However, there is a need for a simple, efficient means ofseparating liquid phases during fluorous phase synthesis of organicmolecules, particularly a method amenable to use with automated methodsfor such syntheses.

Furthermore, complete removal of the liquid from the multiplicity ofvessels by spinning the array of wells attached with “reverse tilt”(tilting away from the axis of rotation) can find its application inbiological assays where fast repeated washing of surface bound reagentsor cells are required, and in applications where synthesis is donedirectly on the surface of the reaction vessels.

SUMMARY OF THE INVENTION

In accordance with the above objects, the present invention providesmethods for elimination of a liquid phase from reaction vesselscomprising positioning a plurality of reaction vessels containing aliquid or mixture of liquids in a holder on the perimeter of acentrifuge rotor. The holder, and thus the reaction vessels, are held ina tilted position with a tilt away from the axis of rotation. The rotoris then spun at a speed that expels the liquid from the vessels. Thismethod of elimination can be used during solid-phase organic synthesis,for example synthesis of peptides or nucleic acids. Optionally, thesesteps can be repeated. Similarly, the reaction vessels may be containedin a microtiter plate or the reaction vessel may be a microtiter plate.

The expelled liquid can be collected in a collection pocket in theholder having a volume sufficient to collect and retain any liquidexpelled from the vessels, or can be collected in a waste reservoir inor outside of the centrifuge.

In an additional aspect, the invention provides methods of synthesis ofcompounds comprising providing a reaction vessel containing a firstbuilding block coupled to the vessel itself. The vessel is thenpositioned in a holder on the perimeter of a centrifuge rotor. A secondbuilding block is added to the vessel under conditions that allow thecoupling of the first and second building blocks, and the rotor is spunat a speed sufficient to expel the liquid from the vessel. Optionaladditional steps of repeating the procedure or washing steps can also beincluded. The building blocks can include amino acids and nucleosides.

In a further aspect, the Invention provides methods for separating atleast two immiscible or partially miscible liquids comprisingpositioning a plurality of reaction vessels containing the liquids in aholder on the perimeter of a centrifuge rotor. The rotor is then spun ata speed such that the lower layer of the multiphase system is retainedin a “pocket” of the vessels and the upper layer is expelled from thevessels.

In an additional aspect, the invention provides apparatus comprising acentrifuge comprising a rotor designed to hold reaction vessels at atilt away from the axis of rotation and a waste reservoir connected tothe centrifuge to hold liquids expelled from the reaction vessels. Inone embodiment, the waste reservoir is connected to the centrifuge witha tube. The apparatus may optionally comprise a liquid distributionsystem and a computer processor.

BRIEF DESCRIPTION OF THE FIGURES

The present invention can be understood more completely by reference tothe following detailed description, examples, appended claims andaccompanying figures.

FIGS. 1A, 1B and 1C illustrate separation of two immiscible or partiallymiscible liquid phases in a “pocket” of the vessels and expulsion ofupper liquid layer achieved according to the method of the invention.FIG. 1A illustrates the lower liquid phase and upper liquid phase in thevessel prior to centrifugation. FIGS. 1B and 1C illustrate the pocketcontaining retained lower liquid phase layer during spinning (andremoval of the upper liquid layer).

FIG. 2 illustrates the path of liquid removed from a vessel, such as awell of a microtiter plate by centrifugation. The straight lip at theupper end of each well of the microtiter plate prevents the liquid fromentering the well closer to the edge of a centrifugal plate—this well ishigher and the lip wall is tilted in the direction to the bottom of theplate. The large arrow represents the vector resulting from centrifugaland gravitational forces. The small arrow with thin trailing lineillustrates the direction of the flow of liquid removed.

FIGS. 3A and 3B illustrate an alternative embodiment of the invention inwhich a vessel having a lip facing inward when spun according to themethod of the invention “creates” a “pocket” in which the lower liquidphase is retained.

FIGS. 4A, 4B and 4C illustrate the situation in which wells are tiltedin “reverse” tilt and no “pocket” is as formed during centrifugation.The result is the complete removal of all liquid from the wells.

FIG. 5 shows the UV spectra of wells before and after two steps ofparallel extraction proving complete elimination of contamination byaromatic hydrocarbon.

FIG. 6 shows a cross section of a centrifuge with a waste reservoirconnected at the bottom of the centrifuge and with the rotor holding areaction vessel at a fixed position titled away from the axis ofrotation.

DETAILED DESCRIPTION OF THE INVENTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of this invention is presented herein with respect tofigures that illustrate preferred embodiments of elements of thisinvention. However, this invention includes those alternativeembodiments of these elements performing similar functions in similarmanners that will be apparent to one skilled In the art from theentirety of the disclosure provided. In addition, all referencesdisclosed herein are incorporated by reference in their entirety.

The present invention is based on a discovery of a simple efficientmeans for separation of two (or more) immiscible, or partially miscibleliquids of different density, e.g., removal of upper layer or layers ofliquid from lower layer, used for parallel extraction and/or in fluorousphase organic syntheses. In one embodiment of the invention, thefluorous phase organic synthetic protocol utilizes widely available,disposable reaction vessel arrays, such as microtiter style plates. Inan alternative embodiment of the invention, the synthetic protocolutilizes a vessel with a lip facing inward (see FIG. 2) spun around itsaxis to create a “pocket” in which the lower layer of the multiphasesystem is retained. According to the present invention, however, anyvessel or array of vessels or plurality of arrays of vessels which canbe placed in a tilted position on the perimeter of a centrifuge, can beused in the method of the invention. The method of the invention forseparating of two (or more) immiscible, or partially miscible liquids ofdifferent density during a parallel extraction and/or in fluorous phaseorganic synthetic process comprises: (1) positioning a reaction vesselor an array of reaction vessels, such as a microtiter plate having anarray of reaction wells, said vessel(s) containing a multilayer systemof liquid phases, on the perimeter of a centrifuge rotor in a tiltedposition; and (2) spinning the rotor of the centrifuge at a speed sothat the lower layer fills a “pocket” of the vessels and the upperliquid phases are expelled from the vessels. In one embodiment of theinvention, the rotor is spun at a speed so that the centrifugal force onthe radius corresponding to the reaction vessels which are closest tothe axis of rotation is significantly greater than the force of gravity,and the lower layer forms a “pocket” of the vessels and the upper layersare expelled from the vessels. The volume of a “pocket” is determinedby: (i) the degree of the tilt, (ii) the speed of rotation, and (iii)the distance of the particular reaction vessel from the axis ofrotation. The appropriate combination of these factors determines thevolume of residual liquid retained in the pocket and thereforecompleteness of upper layer removal. However, since it is desired thatall reaction vessels in a multivessel arrangement of a reaction block(such as a microtiter plate) should undergo the removal of the upperlayers of liquid to the same degree, it is important that the angle ofthe liquid surface in the “pocket” of the reaction vessels during thecentrifugation is as close to 90 degrees with respect to the center ofrotation as possible. In one embodiment, the removed liquid phase iscollected on the wall of the centrifuge. In an alternative embodiment,the removed liquid phase is collected in a “collecting pocket” or aseries of “collecting pockets” attached at the perimeter of thecentrifuge rotor. The apparatus of the invention comprises a holderadapted to attaching a reaction vessel or an array of reaction vessels,e.g., a microtiter plate, to a rotor of a centrifuge, said holdercomprising one or more indentations or groves designated “collectingpockets” positioned along one side of said holder said collectingpockets having a volume sufficient to collect and retain any liquidexpelled from the reaction vessels, e.g./ the wells of the microtiterplate, when the holder and attached reaction vessels are spun by thecentrifuge rotor. According to the invention, the holder can hold asingle or individual microtiter plate or a plurality of microtiterplates, each plate comprising an array of vessels. One or more of theholders can be attached to the rotor of a centrifuge. In anotherembodiment, the apparatus of the invention is an automated integratedapparatus or system for parallel extraction and/or for fluorous phasechemical synthesis, comprising: (a) a centrifuge in which an array ofreaction vessels suitable for parallel extraction and/or for fluorousphase organic synthesis can be spun in a tilted position; (b) a liquiddistribution device; and (c) a computer for processing a program ofinstructions for addition of liquid phase to and removal, viacentrifugation, of upper layer liquid phase from the reaction vesselsaccording to the program.

In general, the methods and apparatus of the invention find use incombinatorial chemical synthesis. By way of introduction, combinatorialchemistry synthesis protocols prescribe the stepwise, sequentialaddition of building blocks to intermediate and/or partially synthesizedintermediate compounds in order to synthesize a final compound. Insolid-phase synthesis, final compounds are synthesized attached tosolid-phase supports that permit the use of simple mechanical means toseparate intermediate, partially-synthesized intermediate compoundsbetween synthetic steps. Typical solid-phase supports include beads,including microbeads, of 30 microns to 300 microns in diameter, whichare functionalized in order to covalently attach intermediate compounds(or final compounds), and made of, e.g., various glasses, plastics, orresins.

WO 99/25470, hereby incorporated by reference, describes the use of acentrifuge in solid-phase synthetic reactions, wherein particles ofsolid phase (microbeads) are contained in reaction vessels such asmicrotiter plates. Synthesis is achieved by the stepwise addition of“building blocks” of the biopolymer, followed by centrifugation thatdrives the liquid phase out of the reaction vessels yet traps the solidphase in “pockets”.

This general idea can be applied to differential phase syntheticreactions as well. While described for fluorous synthesis, one of skillin the art will appreciate that these techniques work for other phasedependent synthetic methods as well.

The principle of fluorous phase synthesis is very similar to solid phasesynthesis. In fluorous phase synthesis one of the reagents is attachedto a high fluorine content block (“fluorous tail”), which assures thatthis reactant will always have a tendency to stay in fluorocarbon basedsolvent layer. Due to the fact that some fluorocarbon based solvents arenot miscible (or only partially miscible) with both organic solvents andwater and that this phase is in most cases the phase with the highestdensity, its properties can be used to mimic the solid phase principleof synthesis. Due to the fact that fluorous phase synthesis technologyis at its very early stage of development, the general process forapplication in the combinatorial synthesis can be only speculated on.Fluorous phase combinatorial synthesis should proceed according to thefollowing steps. In a first step, reaction vessels are charged with afluorous phase, e.g. benzotrifluoride, and the first component of thesynthesis (sometimes referred to herein as “the first buildingblock”)with attached “fluorous tail” (block containing high proportionof fluorine atoms) is delivered to all wells. Subsequently, a pluralityof building block addition steps are performed, all of which involverepetitive execution of the following substeps, and in a sequence chosento synthesize the desired compound. First, a sufficient quantity of asolution containing the building block moiety (e.g. the “second buildingblock”, “third building block”, etc.) selected for addition isaccurately added to the reaction vessels so that the building blockmoiety is present in a molar excess to the intermediate compound(compound with fluorous tail). The reaction is triggered and promoted byactivating reagents and other reagents and solvents, which are alsoadded to the reaction vessel. The reaction vessel is then incubated at acontrolled temperature for a time, typically between 5 minutes and 24hours, sufficient for the building block addition reaction ortransformation to go to substantial completion. Optionally, during thisincubation, the reaction vessel can be intermittently agitated orstirred. Finally, in a last substep of building block addition, thereaction vessel containing the fluorous phase with intermediate compoundattached to fluorous tail is prepared for addition of the next buildingblock by removing the reaction fluid and thorough washing andreconditioning the fluorous phase by washing (repetitive addition andremoval by centrifugation) with water and/or organic solvents. Thelimitation is that the fluorous phase must form always the lower phaseof the system, which can be multilayer (multiphase).

Washing typically involves three to seven cycles of adding and removinga wash solvent. Optionally, during the addition steps, multiple buildingblocks can be added to one reaction vessel in order to synthesize amixture of compound intermediates attached to one fluorous tail, oralternatively, the contents of separate reaction vessels can be combinedand partitioned in order that multiple compounds can be synthesized inone reaction vessel.

After the desired number of building block addition steps, the finalcompound is present in the reaction vessel attached to the fluoroustail. The final compounds can be utilized either directly attached tothe fluorous tail, or alternatively, can be cleaved from the fluoroustail and purified by extraction. Examples of fluorous phase syntheticprotocols can be found in the following references: Curran, D. P. (1996)Combinatorial organic synthesis and phase separation: Back to thefuture. Chemtracts:Org. Chem., 9, 75-87; Curran, D. P., & Hoshino, M.(1996) Stille couplings with fluorous tin reactants: Attractive featuresfor preparative organic synthesis and liquid-phase combinatorialsynthesis. J. Org. Chem., 61, 6480-6481; Curran, D. P. (1998) Fluoroussynthesis: An alternative to organic synthesis and solid phase synthesisfor the preparation of small organic molecules. Canc. J. Sci. Amer., 4,S73S76; Curran, D. P. (1998) Strategy-level separations in organicsynthesis: From planning to practice. Angew. Chem. Int. Ed., 37,1175-1196: Ogawa, A., & Curran, D. P. (1997) Benzotrifluoride: A usefulalternative solvent for organic reactions currently conducted indichloromethane and related solvents. J. Org. Chem., 62, 450-451;Studer, A., Jeger, P., Wipf, P., & Curran, D. P. (1997) Fluoroussynthesis: Fluorous protocols for the Ugi and Biginelli multicomponentcondensations. J. Org. Chem., 62, 2917-2924; Studer, A., & Curran, D. P.(1997) A strategic alternative to solid phase synthesis: Preparation ofa small isoxazoline library by “fluorous synthesis”. Tetrahedron, 53,6681-6696; Studer, A., Hadida, S., Ferritto, R., Kim, S. Y., Jeger, P.,Wipf, P., & Curran, D. P. (1997) Fluorous synthesis: A fluorous-phasestrategy for improving separation efficiency in organic synthesis.Science, 275, 823-826. As for all the references herein, these areexpressly incorporated by reference in their entirety.

Accordingly, in a preferred embodiment the invention provides methodsfor separation of immiscible or partially miscible liquid phases ofdifferent density during a parallel extraction, including a fluorousphase organic synthetic process. The methods comprise: (1) positioning areaction vessel or one or more arrays of reaction vessels, such as oneor more microtiter plates, said vessels containing an immiscible orpartially miscible liquid phases of different density on the perimeterof a centrifuge rotor in a tilted position; and (2) spinning the rotorof the centrifuge at a speed so that the lower phase is retained in a“pocket” of the vessels and the upper liquid phase(s) is (are) expelledfrom the vessels. In the case where only one row of vessels is placed atthe perimeter of the centrifuge rotor, the ratio of centrifugal forceversus gravitation determines the volume of the “pocket” used for theseparation of liquid phases in all vessels and even very low ratio (suchas 1:1) can be successfully used. The important factor is only thereproducibility of the speed of centrifugation.

In one embodiment of the invention, the rotor of the centrifuge is spunat a speed so that the centrifugal force on the radius corresponding tothe reaction vessels which are closest to the axis of rotation issignificantly greater than the force of gravity so that the lower liquidphase is retained in a “pocket” of the vessels and the upper liquidphase(s) is (are) expelled from the vessels. The volume of a “pocket” isdetermined by: (i) the degree of the bit, (ii) the speed of rotation,and (iii) the distance of the particular reaction vessel from the axisof rotation. Since it is desired that all reaction vessels in amultivessel arrangement or array of vessels (such as a microtiter plate)should undergo the removal of the upper liquid phase to the same degree,it is important that the angle of the liquid surface in the “pocket” ofthe reaction vessels during the centrifugation is as close to 90 degreeswith respect to the axis of rotation as possible. As used in the presentapplication, the term “significantly greater than the force of gravity”is intended to mean that the force is at least about 5 to 300×G,preferably about 10 to 300×G, and even more preferably about 100 to300×G. In other words, the centrifuge is spun at a speed so that theratio of the centrifugal force to gravity, i.e., the RelativeCentrifugal Force (RCF) is at least about 5 to 300, preferably about 10to 300, and more preferably about 100 to 300. Values of RCFsignificantly greater than 1 are required if individual vessels areplaced at different distances from the center of rotation. To achieveuniform distribution of liquid in all vessels it is important to removeas much as possible of the upper liquid phase from all wells. Thetheoretical value of an angle of liquid surface achievable in thecentrifuge versus liquid in non-disturbed state is 90 degrees. Thisrequires a value of the above mentioned ratio (RCF) reaching infinity.For practical reasons, the difference between 89 degrees (ratio 100:1)or 85 degrees (ratio 18:1) may be acceptable. Acceptability of thisvalue depends on the degree of the tilt determining the absolute valueof the “pocket” volume. The greater the tilt, the bigger the “pocket”volume, and the bigger the tolerance to the different ratio values atdifferent radiuses. The maximal possible value of the tilt in “fixedtilt” centrifuges is 45 degrees, however, this bit is completelyimpractical because the maximal volume of liquid in the well is equal tothe volume of the theoretical “pocket”.

Higher tilt is possible in the case of “dynamically adjustable tilt”centrifuges (centrifuges in which plate is horizontal in standstillstate and “swings our” to a limited position during rotation). Accordingto one mode of one embodiment of the method of the invention, when thereaction vessels used are one or more arrays of regular wells in amicrotiter plate, the rotor of the centrifuge is spun at a speed so thatthe centrifugal force on the radius of wells closest to the axis ofrotation is about 5 to 300×G. preferably about 10 to 300×G, and morepreferably about 100 to 300×G; and the angle of bit of the plate isabout 1 to 45, preferably 5 to 20, and more preferably 5 to 15 degrees.According to another mode of this embodiment of the method of theinvention, when the reaction vessels used are one or more arrays ofmicrowells in a microtiter plate, the rotor of the centrifuge is spun ata speed so that the centrifugal force on the radius of wells closest tothe axis of rotation is about 5 to 300×G, preferably about 10 to 300×G,and more preferably about 100 to 300×G and the angle of tilt of theplate is about 2 to 25, preferably 2 to 10 degrees. In one embodiment,the upper liquid phase is collected on the wall of the centrifuge. In analternative embodiment, the upper liquid phase is collected in a“collecting pocket” or a series of “collecting pockets”. FIG. 1illustrate retention of lower liquid layer in a “pocket” of the vesselsand expulsion of upper liquid layer achieved according to the method ofthe invention. FIG. 2 illustrates the path of upper liquid layer removedfrom a vessel, such as a well of a microtiter plate by centrifugation.The straight lip at the upper end of each well of the microtiter plateprevents the liquid from entering the well closer to the edge of acentrifugal plate—this well is higher and the lip wall is tilted in thedirection to the bottom of the plate. The large arrow represents thevector resulting from centrifugal and gravitational forces. The smallarrow with thin trailing line illustrates the direction of the flow ofliquid removed from the reaction vessels. FIG. 3 illustrates analternative embodiment of the invention in which a vessel having a lipfacing inward when spun according to the method of the invention“creates” a “pocket” in which the lower liquid phase is retained. Theleft portion of FIG. 1 illustrates the lower liquid phase and upperliquid phase in the vessel prior to centrifugation. The right portion ofFIG. 1 illustrates the pocket containing retained lower liquid phaselayer during spinning (and removal of the upper liquid layer). Asdetailed above, a single reaction vessel, a single microtiter plate or aplurality of microtiter plates can be used in the process of the presentinvention.

In addition to differential phase synthesis, the present invention findsuse in “reverse tilt” synthesis reactions. In this embodiment, thereaction vessels, for example in a microtiter plate format, are tiltedin the direction away from the axis of rotation: that is, the open endof the reaction vessel is pointed away from the axis of rotation. Thenegative (or “reverse”) tilt (tilt in the direction away from the axisof rotation) allows for the removal of all liquid content of the well.This may be done for a variety of reasons. In a first embodiment, thismay be applicable for washing of wells, e.g. in biological assays whenbinding to the surface (or modified surface) is studied and removal ofthe excess of the reagents by repetitive washing is required.

A preferred embodiment for the use of “reverse” bit is the situationwherein the synthesis is performed on material firmly attached to thewell; that is, the material will not be expelled from the reactionvessel under the centrifugation conditions used in the process. In apreferred embodiment, the material can be a “tea-bag” type of materialfilled with the solid support on which the synthesis is done, or atextile like material; for examples of these supports see U.S. Pat. No.5,202,418, hereby expressly incorporated by reference.

In a preferred embodiment, the synthesis is performed on one or moremodified surface(s) of the reaction vessel itself. In this embodiment,rather than use a solid support such as a microbead or a dense phase asthe support for a synthetic reaction, the actual surface of the reactionvessel is used as the solid phase for synthetic reactions; liquidreagents are added, reacted, and then the residual liquid is removed viacentrifugation. That is, the reaction vessel, such as a microtiterplate, may be functionalized as a solid support for the synthesis. Inthis embodiment, the reaction vessel may be any material that can bemodified to allow synthesis; possible materials for substrates include,but are not limited to, glass and modified or functionalized glass,plastics (including acrylics, polystyrene and copolymers of styrene andother materials, polypropylene, polyethylene, polybutylene,polyurethanes, TeflonJ, etc.), polysaccharides, nylon or nitrocellulose,resins, silica or silica-based materials including silicon and modifiedsilicon, carbon, metals, inorganic glasses, and plastics, and a varietyof other polymers.

The functionalization of solid support surfaces such as certain polymerswith chemically reactive groups such as thiols, amines, carboxyls, etc.is generally known in the art. Some examples of these surfacechemistries for subsequent addition of building blocks include, but arenot limited to, amino groups including aliphatic and aromatic amines,carboxylic acids, aldehydes, amides, chloromethyl groups, hydrazide,hydroxyl groups, sulfonates and sulfates.

These functional groups can be used to add any number of differentbuilding block moieties to the vessels, generally using knownchemistries, including, but not limited to the use ofamino-functionalized supports, sulfhydryl linkers, etc. There are anumber of sulfhydryl reactive linkers known in the art such as SPDP,maleimides, a-haloacetyls, and pyridyl disulfides (see for example the1994 Pierce Chemical Company catalog, technical section oncross-linkers, pages 155-200, incorporated herein by reference).Similarly, amino groups on the building blocks and on the surface can beattached using linkers; for example, a large number of stablebifunctional groups are well known in the art, includinghomobifunctional and heterobifunctional linkers (see Pierce Catalog andHandbook, pages 155-200). In an additional embodiment, carboxyl groups(either from the surface or from the building block) may be derivatizedusing well known linkers (see the Pierce catalog). For example,carbodiimides activate carboxyl groups for attack by good nucleophilessuch as amines (see Torchilin et al., Critical Rev. Therapeutic DrugCarrier Systems, 7(4):275308 (1991), expressly incorporated herein). Inaddition, preferred methods include systems that allow post-synthesiscleavage from the reaction vessels.

As will be appreciated by those in the art, the functionalization willdepend on the synthesis done, as outlined below.

As will be appreciated by those in the art, in the reverse tiltembodiments, virtually any solid phase synthesis reaction may be done.Preferred embodiments include organic syntheses, including, but notlimited to, peptide synthesis, nucleic acid synthesis, and smallmolecule synthesis.

In a preferred embodiment, peptides are synthesized. By “peptide” hereinis meant at least two amino acids joined via a peptide bond. The peptidemay be made up of naturally occurring amino acids and peptide bonds, orsynthetic peptidomimetic structures. The side chains may be in eitherthe (R) or the (S) configuration. In the preferred embodiment, the aminoacids are in the (S) or L-configuration. If non-naturally occurring sidechains are used, non-amino acid substituents may be used, for example toprevent or retard in vivo degradations.

The stepwise solid phase synthesis of peptides is well known. Anexemplary solid-phase combinatorial protocol is that for the synthesisof peptides attached to polymer resin, which proceeds according to Lamet al., 1991, Nature 354:82-84; U.S. Pat. No. 5,510,240; Lam et al.,1994, Selective technology: Bead-binding screening. Methods: A Companionto Methods in Enzymology 6:372-380. Another exemplary protocol is thatfor the synthesis of benzodiazepine moieties, which proceeds accordingto Bunin et al., 1992, J. Amer. Chem. Soc., 114:10997-10998 and U.S.Pat. No. 5,288,514. Also, for protocols for the addition ofN-substituted glycines to form peptides, see, e.g., Simon, et al., 1992,Proc. Natl. Acad. Sci. USA, 89:9367-9371; Zuckermann et al., 1992, J.Amer. Chem. Soc., 114:10646-10647; WO PCT94/06,451 to Moos et al.;Approaches for synthesis of small molecular libraries were recentlyreviewed by, e.g., Krchnak and LebI, 1996, Molecular Diversity,1:193-216; Ellman, 1996, Account. Chem. Res., 29:132-143; Armstrong etal., 1996, Account. Chem. Res., 29:123-131.; Fruchtel et al., 1996,Angew. Chem. Int. Ed., 35:1742; Thompson et al., 1996, Chem. Rev.,96:555-600; Rinnova et al., 1996, Collect. Czech. Chem. Commun., 61:171-231; Hemikens et al., 1996, Tetrahedron, 52:45274554. Exemplarybuilding blocks and reagents are amino acids, nucleosides, other organicacids, aldehydes, alcohols, and so forth, as well as bifunctionalcompounds, such as those given in Krchnak and Lebl, 1996, MolecularDiversity, 1:193-216.

In a preferred embodiment, the methods and compositions of the inventionare used to synthesize nucleic acids. By nucleic “acid” or“oligonucleotide” or grammatical equivalents herein means at least twonucleotides covalently linked together. A nucleic acid of the presentinvention will generally contain phosphodiester bonds, although in somecases, as outlined below, nucleic acid analogs are included that mayhave alternate backbones, comprising, for example, phosphoramide(Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein;Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J.Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487(1986); Sawai et al, Chem. Lett 805 (1984), Letsingeret al., J. Am.Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:14191986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (seeEckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press), and peptide nucleic acid backbones and linkages (seeEgholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed.Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al.,Nature 380:207 (1996), all of which are incorporated by reference).Other analog nucleic acids include those with positive backbones (Denpcyet al., Proc. Nati. Acad. Sci. USA 92:6097 (1995); non-ionic backbones(U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423(1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsingeret al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnonribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocycdic sugarsare also included within the definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp169-176). Several nucleic acid analogs aredescribed in Rawls, C & E News Jun. 2, 1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribosephosphate backbone may be done to increasethe stability and half-life of such molecules in physiologicalenvironments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made;alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made.

The nucleic acids may contain any combination of deoxyribo- andribo-nucleotides, and any combination of bases, both naturally occurringand synthetic, including uracil, adenine, thymine, cytosine, guanine,inosine, xanthine, hypoxanthine, isocytosine, isoguanine, etc. Apreferred embodiment utilizes isocytosine and isoguanine in nucleicacids designed to be complementary to other probes, rather than targetsequences, as this reduces non-specific hybridization, as is generallydescribed in U.S. Pat. No. 5,681,702. As used herein, the term“nucleoside” includes nucleotides as well as nucleoside and nucleotideanalogs, and modified nucleosides such as amino modified nucleosides orphosphoramidite nucleosides. In addition, “nucleoside” includesnon-naturally occurring analog structures. Thus for example theindividual units of a peptide nucleic acid, each containing a base, arereferred to herein as a nucleoside.

The stepwise synthesis of nucleic acids is well known, and generallyinvolves the stepwise addition of protected, activated nucleosidemonomers to a solid support, followed by deprotection steps and washingsteps. See generally Gait, Oligonucleotide Synthesis: A PracticalApproach, IRL Press, Oxford, UK 1984; incorporated by reference. This isgenerally done either with phosphoramidite or H-phosphonate nucleosides.This is generally done in one of two ways. First, the 5′ position of theribose is protected with 4′,4-dimethoxytrityl (DMT) followed by reactionwith either 2-cyanoethoxy-bis-diisopropylaminophosphine in the presenceof diisopropylammonium tetrazolide, or by reaction withchlorodiisopropylamino 2′-cyanoethyeoxyphosphine, to give thephosphoramidite as is known in the art; although other techniques may beused as will be appreciated by those in the art. See Gait, supra;Caruthers, Science 230:281 (1985), both of which are expresslyincorporated herein by reference.

In a preferred embodiment, the reverse tilt method is used to synthesizesmall organic molecules. As will be appreciated by those in the art, theliterature contains numerous examples of the synthesis of a variety ofsmall molecules, particularly libraries of small molecules onsolid-phase supports; see for example Pavia et al. Bioorganic &Medicinal Chemistry 1996 4(5):659-666; Liskamp et al., Bioorganic &Medicinal Chemistry 1996 4(5):667-672; Tong et al., Bioorganic &Medicinal Chemistry 1996 4(5):693698; Houghten et al., Bioorganic &Medicinal Chemistry 1996 4(5):709-715, Freier et al., Bioorganic &Medicinal Chemistry 1996 4(5):717-725; Bolton et al., TetrahedronLetters 1996 37(20) 3433-3436, all of which are hereby expresslyincorporated by reference.

In addition, for all the reverse tilt embodiments herein, it may bedesirable to use linkers to attach the first building blocks to thesurface.

Accordingly, the invention provides methods of synthesis using reversetilt centrifugation. In this embodiment, a reaction vessel or array ofvessels are provided that comprise either a pre-functionalized firstbuilding block or the chemistry to attach the first building block ofthe molecule to be made. Subsequently, a plurality of building blockaddition steps are performed, all of which involve repetitive executionof the following substeps, and in a sequence chosen to synthesize thedesired compound. First a sufficient quantity of a solution containingthe building block moiety selected for addition is accurately added tothe reaction vessels so that the building block moiety is present in amolar excess to the intermediate compound. The reaction is triggered andpromoted by activating reagents and other reagents and solvents asneeded, which are also added to the reaction vessel. The reaction vesselis then incubated at a controlled temperature for a time, typicallybetween 5 minutes and 24 hours, sufficient for the building blockaddition reaction or transformation to go to substantial completion.Optionally, during this incubation, the reaction vessel can beintermittently agitated or stirred. Finally, in a last substep ofbuilding block addition, the reaction vessel is prepared for addition ofthe next building block by removing the reaction fluid using the“reverse tilt” centrifugation steps outlined herein and thorough washingand reconditioning as needed. Washing typically involved three to sevencycles of adding and removing a wash solvent. Optionally, during theadditon steps, multiple building blocks can be added to one reactionvessel in order to synthesize a mixture of compound intermediatesattached to one reaction vessel. After the desired number of buildingblock addition steps, the final compound is present in the reactionvessel. It can then be optionally cleaved from the reaction vesselsupport; alternatively, the reaction vessels themselves can be used insubsequent reactions. A variety of exemplary reactions are outlined inW099/25470, hereby incorporated by reference, and include reactions forpeptide and synthetic peptides, benzodiazepine and derivatives,peptoids, N-substituted polyamide monomers. Exemplary building blocksand reagents are amino acids, nucleic acids, other organic acids,aldehydes, alcohols, and so forth, as well as bifunctional compounds.

The present invention also provides apparatus for organic synthesis asoutlined herein. The apparatus of the invention comprise a variety ofcomponents, including a centrifuge and a rotor. In general, the rotorcomprises at least one holder, and preferably a plurality of holders,that each will hold at least a first reaction vessel, and preferably aplurality of reaction vessels.

As will be appreciated by those in the art, the reaction vessels can beconfigured in a variety of ways. In a preferred embodiment, the reactionvessels are in the form of an array of vessels such as a microtiterplate that contains the individual reaction vessels. Particularlypreferred configurations are 96-well and 384-well microtiter plates.

As will be appreciated by those in the art, one of the important aspectsof the invention is that one or more liquid phases are expelled from thereaction vessels upon centrifugation. Accordingly, there are two mainways the system may be configured to allow the collection of theexpelled liquids.

In a preferred embodiment, the holders adapted to attaching a microtiterplate to a centrifuge rotor can have or comprise a series of collectingpockets to collect and retain the liquid expelled from the vesselsduring centrifugation. These collecting pockets can comprise one or moreindentations or grooves having a volume sufficient to collect and retainany expelled liquid.

In an alternative preferred embodiment, the holder does not havecollecting pockets. In the latter situation, the liquid expelled isdeposited on the walls of the centrifuge. In this embodiment, thecentrifuge is configured such that there is a collecting pocket orreservoir, generally in the bottom of the centrifuge, such that gravityflow of the expelled liquids causes the liquids to pool in the pocketThis may be periodically emptied as needed, and can comprise a port orvalve that allows drainage.

Altematively, the centrifuge is configured to have a tube or pipeleading to a waste reservoir; this tube is also generally at the bottomof the centrifuge. The gravity flow of the expelled liquids can thenlead to collection of the waste outside the centrifuge.

In a preferred embodiment, the holder(s) hold the reaction vessels in atilted position. The holder(s) may either hold one or more of thereaction vessels in a fixed tilted position or in a position in whichthe angle of tilt can be changed flexibly.

As outlined herein, the tilt of the rotor can be towards the axis ofrotation, resulting in the retention of a phase of the reaction.Alternatively, when the synthetic reaction is done on material firmlyattached to the reaction vessel (e.g. with a force such that it will notbe expelled during the centrifugation) or when the reaction is done onthe reaction vessel itself, the tilt of the rotor can be away from theaxis of rotation (“reverse tilt”)as described herein.

In a preferred embodiment, each holder contains only one set or array ofreaction vessels. Thus, for example, the holder may contain grooves orrails to position the reaction vessels, e.g. microtiter plate, in theholders. Alternatively, the reaction vessels may be “stacked” or“layered”. However, placing single sets or arrays of reaction vesselssuch as individual microtiter plates on the centrifuge perimeter has anadvantage of simple interfacing with liquid distribution automats (suchas Packard Canberra, Tecan, Hamilton, and others). A liquid distributiondevice can be placed onto the top of a centrifugal synthesizer.Particularly preferred are liquid distribution systems for simultaneousdispensing in a format that fits the reaction vessel configuration; forexample, when the reaction vessels are in the form of a 96 wellmicrotiter plate, the liquid distribution system is preferably a 96channel device.

The liquid distribution system can also comprise a set of reservoirs andtubes for delivery; for example, the liquid distribution system can havea 96 channel liquid distributor that can deliver solvent or solutions ofreagents from different bottles into the plate positioned under theneedles of the distributor. For example, for nucleic acid synthesis,preferred embodiments include separate reagent bottles for eachnucleoside.

In a preferred embodiment, the liquid distribution system is anintegrated system; that is, the liquid is distributed into the reactionvessels when they are present in the centrifuge; the reaction vesselsare not removed from the centrifuge for addition of reagents. Ingeneral, the liquid distribution system is an integral part of thecentrifuge; the liquid is delivered without removing the lid of thecentrifuge.

In addition, the apparatus of the invention can further comprise aprocessor or computer to control the synthesis of the moieties. Forexample, a computer may be used that processes a program of instructionsof stepwise additions of liquid phases, reagents, solvents, washes, etc.to the reaction vessels, followed by centrifugation steps for removal ofliquids from the reaction vessels. Thus, the present invention providesmethods executed by a computer under the control of a program, thecomputer including a memory for storing the program. The program isdirected to the addition of reagents to the reaction vessels using theliquid distribution system, allowing incubation as needed, and removingunreacted reagents and liquid by centrifugation for a defined time at adefined speed, with wash steps and repetition as required.

In addition to the components mentioned above, the centrifuge may alsocomprise additional components. For example, the centrifuge can comprisea sensor to signal the computer and liquid distribution system when aset of reaction vessels in a particular orientation, and a motor torotate the rotor into the correct orientation for liquid delivery, alsoin control of the computer. Furthermore, in the case of adjustable tiltholders or rotors, the centrifuge can utilize a control and a sensor tocontrol the degree of tilt.

The integrated device is useful as a “centrifugation synthesizer” forfluorous phase synthetic processes.

The methods and apparatus of the invention find use in a number ofapplications, as outlined herein.

In a preferred embodiment, the methods and apparatus of the presentinvention are advantageously useful for the manual or automatedpreparation of combinatorial libraries or megaarrays of compounds byfluorous phase organic synthesis. As is well known to those skilled inthe art, such combinatorial libraries or megaarrays have numerous uses,in particular, for the selection of pharmaceutical lead compounds, forthe optimization of pharmaceutical lead compounds and for theidentification and/or isolation of pharmaceutical drugs. The methods andapparatus of the invention for liquid/liquid phase separation can alsoadvantageously be used for parallel extraction and purification ofcompound arrays synthesized or obtained by other methods. Otherapplications in analytical chemistry (extraction, desalting or othermeans of parallel preparations of samples), biochemistry (parallelprocessing of samples) are envisioned.

The use of complete removal of liquid from the arrays of vessels can beapplied in the biological screening where binding to the surface of thevessels (modified surface by attached reagent or cell culture) isinvestigated. In this case the tilt of the vessel during thecentrifugation is “reversed”, i.e. no “pocket” is formed during thecentrifugation. In this case, for example, any number of binding assaysmay be done. For example, ELISA type assays are frequently done in amicrotiter plate format, where antibodies are attached using a varietyof known chemistries. The addition of sample(s) and additional reagentcomponents, with washing as required, may utilize the present invention.Furthermore, in this embodiment, the apparatus may comprise additionalcomponents such as fluorescence readers.

Similarly, reverse tilt reactions can be used in synthetic reactions asoutlined above, with particular emphasis on nucleic acid and peptidesynthesis.

EXAMPLE: REMOVAL OF UPPER LAYER LIQUID PHASE WITHOUT TRANSFER OF LOWERLAYER LIQUID PHASE

Ten percent solution of ethanol in water saturated with toluene wasdistributed into wells of microtiterplate (40 uL per well). Ethylacetate (150 uL) was repeatedly distributed into the wells andmicrotiterplates were shaken for 1 minute and centrifugated in tiltedarrangement. FIG. 5 shows UV spectra of wells before and after two stepsof parallel extraction proving complete elimination of contamination byaromatic hydrocarbon.

1. An apparatus comprising: a) a centrifuge comprising a rotor rotatableabout an axis of rotation which includes at least one reaction vessel ata permanently fixed angle tilted with respect to said axis of rotationso that an open end of said at least one reaction vessel is pointed awayfrom said axis of rotation; and b) a waste reservoir connected to saidcentrifuge to hold liquids expelled from said reaction vessels.
 2. Anapparatus according to claim 1 wherein said waste reservoir is connectedto the bottom of said centrifuge.
 3. An apparatus according to claim 1wherein said waste reservoir is connected with a tube to saidcentrifuge.
 4. An apparatus according to claim 1 wherein said rotorcomprises a plurality of holders, each holder designed to hold at leastone microtiter plate at said permanently fixed angle.
 5. An apparatusaccording to claim 1 further comprising a liquid distribution system. 6.An apparatus according to claim 5 wherein said liquid distributionsystem is integrated into the centrifuge.
 7. An apparatus according toclaim 1 further comprising a computer.
 8. An apparatus according toclaim 1 wherein said at least one reaction vessel further comprises asolid-phase support adapted for solid-phase synthesis.
 9. An apparatusaccording to claim 8 wherein said solid-phase support is firmly attachedto an interior surface of said at least one reaction vessel.
 10. Anapparatus according to claim 8 wherein said solid-phase support is amodified interior surface of said at least one reaction vessel.
 11. Anapparatus according to claim 10 wherein said at least one reactionvessel is a microtiter plate functionalized as a solid-phase support forsaid solid-phase synthesis.